Exchange Protein Directly Activated By CAMP Epac1 Contributes To Post-operative Pain Through P-ERK?VEGF Expression

Objective Study of guanine nucleotide exchange conversion fa ctor protein(exchange proteins directly activated by cAMP,Epac1)in rats with the role and mechanism of persistent postoperative pain.Methods(1)Adult male Sprague-Dawley rats were randomly d ivided into three groups(na?ve group,SMIR group and sham group).Mechanical stimulation were delivered using Up-Down paradigm w ith von Frey filaments.The expression of Epac1 in the L3–5 spinal cord?DRG?incision site muscle of na?ve ?sham?SMIR group were examined by immunofluorescence staining and Western-blot.The cellular localization of Epac1 in spinal cord ?DRG and incisi on site muscle were definited by immunofluorescence double stainin g.(2)Adult male Sprague-Dawley rats were randomly divided into t hree groups(na?ve group,saline group and Epac1 agonist(8-pCPT)g roup).The effect of 8-p CPT on pain was checked by behavioral test ing,and the protein level of(Phosphorylation of extracellular signal-regulated kinase,p-ERK)and(vascular endothelial growth factor,V EGF)were examined by Western-blot.(3)Adult male Sprague-Daw ley rats were randomly divided into three groups(SMIR group,NC group and Epac1-siRNA group).Theffect of 8-pCPT on pain was c hecked by behavioral testing,and the protein level of p-ERK ?VEGF were examined by Western-blot.Results(1)Before SMIR surgery,the basal MWT of the ipsilateral paw was tested for na?ve,sham-operated and SMIR rats and they showed similar threshold.The MWT of SMIR-operated rats compared with the baseline decrease at the postoperative day(P<0.05),peaked at 7-12d(P<0.01)and increased by postoperative day 21(P<0.01).However,no significant difference was observed between the sham and the na?ve groups,and the na?ve groups and sham-operated did not show any significant change at all time points.SMIR treatment caused significant increases in expression of Epac1 in spinal cord?DRG and incision site muscle by the densitometric analysis of Western blot and immunofluorescence staining.Confocal images showed that Epac1 expression was generally confined to(GFAP-positive)astrocytes,with little expression in(NeuN-positive)neurons and colocalized with DRG large neurons(axons)marker NF200,DRG non-peptide neurons marker IB4,DRG peptide neurons marker CGRP,and colocalized with the muscular macrophages marker CD68,but not with the vascular endothelial cells marker VIII.(2)intraplantarly injected 8-pCPT suppresses mechanical allodynia of na?ve rats and significantly increased pERK,VEGF protein levels(P<0.05)in L3-L5 spinal cord and DRG.(3)Intrathecally injected Epac1 siRNA on post-SMIR day 7 reverses mechanical allodynia,protein levels for the pERK,VEGF were significantly decreased(P<0.05)in L3-L5 spinal cord and DRG of Epac1 siRNA-injected rats compared with the NC-injected ratsConclusions SMIR–induced upregulation of the Epac1 may activation of the p-ERK?VEGF plays an important role in promoting post-surgical pain.